The related parasitic protozoa, Trypanosoma brucei and Trypanosoma cruzi cause substantial and economically significant morbidity and mortality throughout the third world. In total about 20 million people are at risk in Africa and South America. Vaccines are not available and drug therapy is either inadequate because of resistance and toxicity, or in the case of chronic infections of T. cruzi, unavailable. The development of new therapeutics requires the discovery of chemical structures which will influence biological activity. Drug development has traditionally been accomplished by random screening of chemical libraries, using biological assays for cell function. More directed methods are being sought and interest has become focused on designing drugs by targeting specific cell enzymes. Initially mechanism-based approaches and more recently, structure-based approaches have been developed. The discovery of the anti-trypanosomal agent alpha-difluoromethylomithine (DFMO), an inhibitor of ornithine decarboxylase (ODC), is a result of mechanism-based design. DFMO has been only partially successful as a drug; however, its usefulness serves to identify polyamine biosynthesis as a target for chemotherapeutic intervention against the trypanosomes. We plan to study the polyamine biosynthetic enzymes from the trypanosomatids, with the goal of determining enzyme structure/function. However, molecular understanding of these enzymes should also illuminate methods to inhibit the parasite, but not the host, enzymes leading to the design of novel drugs. The selected target enzymes are ODC from Trypanosoma brucei and Leishmania donovani, T. cruzi arginine decarboxylase (ADC), which does not have a host analog, and T. cruzi S- adenosylmethionine decarboxylase (SAMDC). The comparative biochemical properties of the analogous host and parasite enzymes will be analyzed, including: 1) enzyme kinetics on various substrate and cofactor derivatives, to determine the structural features of the ligand required for enzyme recognition and catalysis and, 2) site-directed mutagenesis and three-dimensional structure determination to identify the amino acids within the enzyme that bind substrate and catalyze the chemical reaction. Protein modeling will be used to identify residues that account for biochemical differences between the host and parasite enzymes and these predictions will be tested by mutagenesis. Inhibitors will be designed to exploit these differences using structure evaluation and computer-assisted design. These studies should lead to the development of new drug-against an important class of deadly organisms in the context of a kinetic and structural analysis of enzyme function.